Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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1 Electrophoretic Techniques40

1.4.1.1 Titration Curve Analysis

The net charge curves are also called “titration curves”. The titration

curves of proteins can be displayed by a simple method (Rosengren

et al. 1976): A square gel – containing carrier ampholytes, but no sam-

ples – is submitted to an electric field until the carrier ampholytes

form a pH gradient from 3 to 10. Then the gel is rotated by 90

degrees, the sample proteins are pipetted into a groove in the gel

across the pH gradient. Now an electric field is applied perpendicular

to the pH gradient: the carrier ampholytes are uncharged at their pIs

and will not move. But, as shown in Figure 1.10, the proteins will

migrate towards the cathode or the anode according to their charge

sign and mobility, and will thus form titration curves (net charge

curves).

Fig. 1.10: Titration curves of a mixture of pI marker proteins

under native conditions. The steeper the curve, the faster will be

the migration of a protein in IEF. Note that some proteins have

a flatter curve above their pI.

The proteins possess very individually shaped curves also under dena-

turing conditions. Practical experience has shown that more proteins

have a steeper curve below their pI. This is the reason, why mostly

sample application close to the anode results in better pattern.

1.4.1.2 pH Gradients

In practice there are two ways to establish a pH gradient in a gel:

pH gradients which are formed in the electric

field by amphoteric buffers, the carrier ampho-

lytes;

Rosengren A, Bjellqvist B,

Gasparic V. In: Radola BJ,

Graesslin D. Ed. Electro-

focusing and isotachophoresis.

W. de Gruyter, Berlin (1977)

165–171.

1.4 Isoelectric Focusing 41

Immobilized pH gradients in which the buffer-

ing groups are part of the gel medium.

Carrier ampholytes generated pH gradients This was the first devel-

oped technique for isoelectric focusing. Svensson (1961) has designed

the theoretical basis for preparing “natural” pH gradients, while the

practical realization is the work of Vesterberg (1969): the synthesis of

a heterogeneous mixture of isomers of aliphatic oligoamino-oligocar-

boxylic acids. The result is a spectrum of low molecular weight

ampholytes with closely neighboring isoelectric points, and with high

buffering power at their pI.

The general chemical formula is the following:

–CH

–N–(CH

)

–N–CH

–

(CH

)

(CH

)

COOH

R = H or –(CH

)

–COOH, x = 2 or 3

These carrier ampholytes possess the following properties:

High buffering capacity and solubility at the pI;

Good and regular electric conductivity at the pI;

Absence of biological effects;

Low molecular weight.

Naturally occurring ampholytes such as amino acids and peptides

have only low or no buffering capacity at their isoelectric point. They

can therefore not be used.

The pH gradient is established by the electric field. At the begin-

ning, the gel with carrier ampholytes has a uniform mean pH value.

Almost all the carrier ampholytes are charged: those with the higher

pI positively, those with the lower pI negatively.

When an electric field is applied, the negatively charged carrier

ampholytes migrate towards the anode, the positively charged ones to

the cathode, with velocities depending on their net charges. The car-

rier ampholytes align themselves in between the two electrodes

according to their pI and will determine the pH of their environment.

A (relatively) stable and gradually increasing pH gradient results; for

instance from 3 to 10. The carrier ampholytes lose a great part of their

charge, so the conductivity of the gel decreases substantially.

Because these are mixtures of buffers, the gradient profile will

never be perfectly linear. By adding narrow range gradient mixtures

to wide gradient mixtures the final shape of the gradient can be mod-

ified according to special resolution requirements.

Svensson H. Acta Chem Scand

15 (1961) 325–341.

Vesterberg, O. Acta Chem.

Scand 23 (1969) 2653–2666.

Alternative synthesis chemis-

tries have been developed later.

The chemical structures of

other amphoteric buffers are

different. Their function is the

same, but the properties of the

gradients can be different.

By controlling the synthesis and

the use of a suitable mixture

the composition can be moni-

tored for the preparation of

regular and linear gradient.

The anodal end becomes acidic

and the cathodal side basic.

1 Electrophoretic Techniques42

To maintain a gradient as stable as possible, electrode solutions are

applied between the gel and the electrodes, an acid at the anode and a

base at the cathode. Should, for example, an acidic carrier ampholyte

reach the anode, its basic buffering group would acquire a positive

charge from the medium and it would be attracted back by the elec-

tric field.

Isoelectric focusing in carrier ampholytes generated pH gradients

can be performed in various media:

In free liquid phase in capillaries, free flow elec-

trophoresis apparatus and rotating tubing;

In gels made from agarose with very low electro-

endosmosis;

In granulated dextrane gels (Sephadex



);

In polyacrylamide slab gels or individual gel

rods in glass or plastic tubes.

Carrier ampholytes as solvents for proteins

Carrier ampholytes have

another very important function: they help to solubilize proteins,

which stay in solution only in presence of buffering compounds.

Therefore they are also needed for the immobilized pH gradient tech-

nique.

Limitations of carrier ampholyte pH gradients

Isoelectric focusing is a

relatively slow separation method, because proteins have only very

low net charges when they come close to their isoelectric points, and

therefore low mobility. High spatial resolution requires long separa-

tion distance, which causes a long migration time under a high elec-

tric field strength.

Because the carrier ampholytes are in free solution, the gradient

will become instable during long IEF time: the gradient drifts. The

pattern becomes time-dependent and most of the basic proteins are

drifting out of the gel together with the basic part of the gradient.

Another problem: proteins of the sample behave like additional car-

rier ampholytes and modify the profile of the gradient. Thus the gra-

dient shape becomes also sample-dependent.

Immobilized pH gradients These problems are solved with the appli-

cation of immobilized pH gradients, generated by buffering acryla-

mide derivatives copolymerized with the gel matrix (Bjellqvist et al.

1982). The acrylamide derivatives containing the buffering groups are

called Immobilines



. An Immobiline is a weak acid or weak base

defined by its pK value.

Particularly under denaturing

conditions the matrix is highly

viscous leading to slow migra-

tion. Additionally, unfolded

polypeptides migrate slower in

the gel than native proteins.

Carrier ampholyte generated

gradients are also influenced by

the sample load and salt

contents in the sample.

Bjellqvist B, Ek K, Righetti PG,

Gianazza E, Gçrg A, Wester-

meier R, Postel W. J Biochem

Biophys Methods 6 (1982)

317-339.

1.4 Isoelectric Focusing 43

The general structure of an Immobiline is the following:

=CH–C–N–R

II I

R contains either a carboxylic or an amino group.

The acidic substances have dissociation constants in the range

from pK 0.8 to pK 4.6, the basic from pK 6.2 to pK 12. In order to

buffer at precise pH values, at least two different Immobilines are

necessary, an acid and a base. Figure 1.11 shows a diagram of a poly-

acrylamide gel with polymerized Immobilines, the pH value is

defined by the ratio of the Immobilines in the mixture.

Fig. 1.11: Immobilized pH buffer. Schematic drawing of a

polyacrylamide network with co-polymerized buffering groups.

A pH gradient is obtained by continuously varying the ratio of

Immobilines. The principle is that of an acid/base titration, and the

pH value at each stage is defined by the Henderson–Hasselbalch

equation:

pH ¼ pK

þ log

 C

when the buffering Immobiline is a base.

and C

are the molar concentrations of the acid and basic

Immobiline, respectively.

The wider the pH gradient, the

more different Immobiline

homologues are needed.

This concept generates an abso-

lutely continuous pH gradient.

1 Electrophoretic Techniques44

If the buffering Immobiline is an acid, the equation becomes:

pH ¼ pK

þ log

 C

Celentano et al. (1986) and Altland (1990) developed PC programs

for the calculation and simulation of immobilized pH gradients for

the optimization of buffering power and ionic strength distribution.

Practice shows that a pre-calculated pH gradient recipe does not auto-

matically result in a usable IEF gel. Every recipe has to be tested and

optimized with practical experiments.

Further reading There are two books by P.G. Righetti which deal

exclusively with isoelectric focusing in theory and applications.

1.4.2

Preparation of IEF Gels

Carrier ampholyte gels

Casting of such homogeneous polyacryl-

amide gels is very simple: the monomer solution containing the car-

rier ampholytes is mixed with the catalysts and pipetted into a poly-

merization cassette, or into glass or plastic tubes. The carrier ampho-

lyte concentration is mostly 2% (w/v), the gel concentration 4% T and

3% C. To exclude oxygen the solution is carefully overlaid with dis-

tilled water.

The preparation of granulated gels is more intricate, because it is

very important to achieve the optimum liquid content (Westermeier,

2004).

Immobilized pH gradients Immobiline stock solutions with concen-

trations of 0.2 mol/L are used. Two solutions with acrylamide mono-

mers, crosslinker, and catalysts are required to prepare an immobi-

lized pH gradient: one contains the Immobiline cocktail for the acidic

end, the other the cocktail for the basic end. The gel forming mono-

mers are diluted to 4% T and 3% C. The acidic solution is made den-

ser by adding glycerol. The gel is prepared by linear mixing of the two

different monomer solutions with a gradient maker (see Figure 1.12),

like for pore gradients. In principle a concentration gradient is

poured. 0.5 mm thick Immobiline gels, polymerized on a support

film have proved most convenient. During polymerization, the buffer-

ing carboxylic and amino groups covalently bind to the gel matrix.

Because of the low electric conductivity of immobilized pH gradients,

all contaminating compounds, like acrylamide monomers and the

catalysts, have to be removed by washing the gels several times with

deionized water.

Celentano F, Gianazza E, Dossi

G, Righetti PG. Chemometr

Intel Lab Systems 1 (1987)

349–358.

Altland K. Electrophoresis 11

(1990) 140–147.

Righetti PG. In: Work TS, Ed.

Burdon RH. Elsevier Biome-

dical Press, Amsterdam (1983).

Righetti PG. In: Burdon RH,

van Knippenberg PH. Ed. Else-

vier, Amsterdam (1990).

Westermeier R. In Cutler P. Ed.

Protein Purification Protocols.

Second edition. Methods in

Molecular Biology, Volume

244, Humana Press, Totowa,

NJ (2004) 225–232.

1.4 Isoelectric Focusing 45

Fig. 1.12: Preparing an immobilized pH gradient.

Pouring a gradient into a gel cassette.

1.4.3

Isoelectric Focusing in Proteomics

1.4.3.1 IEF Based on Carrier Ampholyte Gradients

Free flow IEF

The free flow electrophoresis apparatus described

above can also be used for isoelectric focusing. Either carrier ampho-

lytes or multi-components buffers, which are mixtures of amphoteric

and non-amphoteric buffers, are added. The latter mixture cannot

develop linear pH gradients, but they reduce the costs. With IEF a

higher number of fractions can be obtained than with the discontinu-

ous field electrophoresis.

Free flow electrophoresis and isoelectric focusing has successfully

been applied for fractionation and proteins in proteomics workflows

(Hoffmann et al. 2001; Obermaier et al. 2005). Lately it has also been

used for the separation of peptide mixtures as a replacement of

strong cation exchange chromatography (Xie et al. 2005). Free flow

IEF is a very reliable and reproducible technique, however good

operator skills and highly sophisticated instrumentation is required.

Liquid phase isoelectric focusing systems Several types of IEF sys-

tems in liquid phase are on the market, which employ membranes

for the separation of the fraction compartments. Temperature differ-

Hoffmann P, Ji H, Moritz RL,

Connolly LM, Frecklington DF,

Layton MJ, Eddes JS, Simpson

RJ. Proteomics 1 (2001) 807–

818.

Obermaier, C, Jankowski V,

Schmutzler C, Bauer J, Wild-

gruber R, Infanger M, Kohrle J,

Krause E, Weber G, Grimm D.

Electrophoresis 26 (2005)

2109–2116.

Xie H, Bandhakavi S, Griffin

TJ. Anal Chem 77 (2005)

3198–3207.

1 Electrophoretic Techniques46

ences and sticking of proteins are tried to be avoided either by con-

tinuously streaming fluids or rotation. The instrumental efforts are

less than with free flow apparatus, but there is always the danger that

hydrophobic proteins stick to membranes.

IEF in a granulated gel bed Gçrg et al. (2002) have developed a very

quick, reproducible and efficient pre-fractionation method: pre-

separation of complex protein mixtures by carrier ampholyte IEF in a

granulated gel bed. The procedure is performed on standard flatbed

electrophoresis equipment at an exactly adjusted temperature of

20 C. The technique is simple: the sample is mixed with low electro-

endosmosis Sephadex, which has been reswollen with rehydration

solution containing urea, CHAPS, DTT and carrier ampholytes. For

the visualization of pH value zones after IEF a mixture of non-protein

amphoteric dyes is added. The slurry is pipetted into a trough and

covered with a thin layer of silicone oil. After 4 hours the proteins are

focused at their isoelectric points. The pH gradient can be deter-

mined by interpolation between the colored marker zones. As shown

in Figure 1.13, the pH fractions are removed with a spatula and

directly applied onto rehydrated IPG strips. In the electric field the

proteins migrate out from the granulated gel into the polyacrylamide

gel of the IPG strip. The fractions can also be stored in a freezer at

–70 C.

This technique has several advantages:

Standard laboratory equipment is used.

The technique is easy to carry out.

The separation is quick.

Denaturing sample pre-fractionation according

to pI in presence of urea, detergents, etc.

No protein losses.

It can be done with high and low protein

amounts

It is also desalting the sample – no microdialysis

is required.

Subsequent separations of the pH fractions in the respective narrow

pH interval IPG strips show a considerable increase in spot number.

Many more of the lower expressed proteins can be detected in this

way.

Gçrg A, Boguth G, Kçpf A, Reil

G, Parlar H, Weiss W. Proteo-

mics 2 (2002) 1652–1657.

1.4 Isoelectric Focusing 47

Fig. 1.13: Pre-fractionation of a complex protein mixture with

isoelectric focusing in Sephadex.

Isoelectric focusing in gel rods for 2-D electrophoresis In the original

high resolution 2-D electrophoresis method the first dimension is car-

ried out in thin gel rods in glass or plastic tubes. The history of this

technique goes back to a paper in German language by Stegemann

(1970), combining isoelectric focusing (IEF) and SDS polyacrylamide

gel electrophoresis. The resolution of 2-D electrophoresis was consid-

erably increased by the introduction of denaturing conditions during

sample preparation and isoelectric focusing by O’Farrell in the year

1975. With this modification the method gained a wide acceptance.

The gel rods contain urea, detergent, reductant and carrier ampho-

lytes to form the pH gradient in the electric field. Usually the sample

is loaded onto the cathodal side of the gel rod, which becomes the

basic end of the gradient in the electric field. This “O’Farrell” techni-

que has been used for about two decades – and is still used by some

laboratories – without major modifications. The potential of the

method for a systematic approach to create a protein database had

been recognized very soon. Anderson and Anderson (1978) have

designed instruments and operation procedures to prepare and run

multiple 2-D gels under the most reproducible conditions, in order to

develop a “Human protein index” (Anderson and Anderson, 1982).

Gels with about 2020 cm have become standard for an adequate

spatial resolution. If one assumes that up to 100 bands can be

resolved in a 20 cm long one-dimensional gel, a theoretical separation

space of 10,000 proteins could be reached in such a gel. In practice

several thousands of proteins can be detected.

Stegemann H. Angew Chem

82 (1970) 640.

O’Farrell PH. J Biol Chem 250

(1975) 4007–4021.

Anderson NG, Anderson NL.

Anal Biochem 85 (1978)

331–354.

Anderson NG, Anderson NL.

Clin Chem 28 (1982)

739–748.

1 Electrophoretic Techniques48

Due to the high concentration of urea the viscosity in the gel is very

high. The denatured proteins are unfolded and thus highly retarded

by the gel matrix. Therefore long migration times are needed, which

lead to a destabilization of the gradient. The soft, thin, and long gel

rods demand high experimental skill. Between pH 6.7 and 7 there is

very often a lack of buffering power, leading to mechanical instability

of the thin gel rods, and an empty area in the spot pattern. The hand-

ling of the technique was rather cumbersome; in many cases the pat-

terns were not reproducible enough. Besides the influence of the

sample composition also batch to batch variations of the carrier

ampholyte mixtures were the reasons for differences in the profiles of

the pH gradients. Gradient drift with prolonged isoelectric focusing

time lead to losses of almost all basic and some of the acidic proteins.

A remedy had soon been found for the display of basic proteins:

O’Farrell et al. (1977) have introduced a modification of the first

dimension run: NEPHGE (non-equilibrium pH gradient electrophor-

esis). Here the sample is loaded onto the acidic end of the gel and the

proteins are separated while the gradient drifts towards the cathode.

The run is stopped after a defined time period, which is approxi-

mately one third of the regular focusing time. However, due to the

time factor it is hard to achieve a good reproducibility. The proteins

are not focused like in IEF; the resolution is limited by the number of

different carrier ampholyte homologues.

Meanwhile most laboratories prefer to use the technique of IEF in

individual IPG strips according to Gçrg et al. (2000), which will below

be described in detail. In these gels all proteins are strongly focused

at their isoelectric points.

There are indications that it is not always an advantage, when pro-

teins are tightly focused at their isoelectric points. High molecular

weight and hydrophobic proteins aggregate with themselves when

they are too tightly focused, and cannot migrate out from the first

dimension gel anymore. This can explain, why sometimes proteins

are missing in an IPG strip 2-D gel, but they are present after a 2-D

electrophoresis run with the traditional NEPHGE procedure.

Some laboratories employ isoelectric focusing in gel rods and

immobilized pH gradient for 2-D electrophoresis side by side as com-

plementary methods.

1.4.3.2 IEF Based on Immobilized pH Gradients

Immobilized pH gradient strips

Generally it is very useful to run dif-

ferent samples in completely separated individual gels. Immobilized

pH gradient gels are the only electrophoresis slab gels, which do not

show any edge effects during the run, when they are cut into strips.

The gels are cut after they have been dried down on the film. Instruc-

Because of the technical limita-

tions, mainly acidic proteins

could be studied in the past.

2-D electrophoresis had some-

times been seen as a technique,

which produces operator-

dependent results.

O’Farrell PZ, Goodman HM,

O’Farrell PH. Cell 12 (1977)

1133–1142.

The proteins are not focused,

but stacked between the

different carrier ampho lyte

homologues.

Gçrg A, Obermaier C, Boguth

G, Harder A, Scheibe B, Wild-

gruber R, Weiss W. Electrophor-

esis 21 (2000) 1037–1053.

This observation has been

made particularly with

membrane proteins.

Gçrg A, Weiss W. In Rabilloud

T, Ed. Proteome research: Two-

dimensional gel electrophoresis

and identification methods.

Springer, Berlin Heidelberg

New York (2000) 107–126.

1.4 Isoelectric Focusing 49

tions and pH gradient recipes for preparing immobilized pH gradient

strips in the laboratory can be found in several book chapters: Gçrg

and Weiss (2000 and 2005) and Westermeier (2004). However, this is

a multistep procedure, which needs some expertise and skill.

Much more reproducible results are obtained, when ready-made

IPG strips are used. Commercially produced strips are prepared

according to GMP industry standards, and they are quality controlled.

A wide choice of different gradients and strip lengths is available on

the market. Additional gradients are being developed for achieving

higher resolution.

Gel sizes The commercial strips are 3 mm wide and have to be

reconstituted to the original thickness of 0.5 mm with rehydration

solution prior to IEF. Thicker and wider strips would have a higher

protein loading capacity. This would, however, not be an advantage,

because the SDS polyacrylamide gel of the second dimension would

show overloading effects of proteins and a high detergent back-

ground. In theory this could be compensated by using SDS gels

thicker than the conventional 1 to 1.5 mm. However, staining of such

thick gel slabs would be very time consuming and not allow a high-

throughput approaches.

Strips are available in several different lengths from 7 cm to 24 cm.

In proteomics usually 18 and 24 cm strips are used, because the high-

est possible resolution is required for proteome analysis. Miniformat

strips with 7 cm length are ideal for optimization of the sample pre-

paration method.

When spatial resolution does not have the highest priority, like for

protein identification with western blotting, small gel formats from 7

to 13 cm are sufficient.

pH gradient types One of the advantages of immobilized pH gradi-

ents is the possibility to reproducibly produce linear gradients from

pH 3 to pH 10. However, there are samples with an uneven distribu-

tion of the proteins over the pH gradient from 3 to 10. The non-linear

gradient pH 3–10 is flat in the acidic range to accommodate more dif-

ferent and higher concentrations of acidic proteins. So far the widest

gradient was designed and applied by Gçrg et al. (1999): pH 3–12. For

increased resolution and higher protein loads acidic (pH 4–7, pH

3–7) and basic (pH 6–11, pH 6–9, pH 7–11) gradients are employed.

Narrow intervals with only one or two pH units allow very high pro-

tein loads and excellent spatial resolution. The narrowest intervals, so

called “ultra zoom gels” with 0.5 pH units, have been applied to 2-D

electrophoresis by Hoving et al. (2000).

Gçrg A, Weiss W. In: Cell

Biology. A Laboratory Manual,

Vol. IV, Ch. 23, 3rd edition. (JE

Celis, Ed.), Elsevier Science &

Academic Press (2005) pp.

175–188.

Westermeier R. Electrophoresis

in Practice. WILEY-VCH, Wein-

heim (2004).

It is very annoying, when the

valuable sample is not sepa-

rated well, just because of a

little mistake occurring in the

laboratory.

With miniformat gels results

are obtained after a few hours.

Gçrg A, Obermaier C, Boguth

G, Weiss W. Electrophoresis 20

(1999) 712–717.

Hoving S, Voshol H, van

Oostrum J. Electrophoresis 21

(2000) 2617–2621.